2.10. Western Blotting
Briefly, equal amounts of total cell lysates were blotted onto
polyvinylidene fluoride membranes, incubated with primary antibodies
overnight at 4°C, washed, and incubated with peroxidase-conjugated
secondary antibody for 1 h at 37°C. The primary antibodies against
p-RIP1 (#65746), RIP1 (#33493), p-MLKL (#37333), MLKL (#14993), and
SOD-2 (#13194s) were obtained from Cell Signaling Technology (Danvers,
MA, USA). RIP3 (ab62344), GCLC (ab53179), Foxo3 (ab47285), GCLM
(ab153967), HO1 (ab68477), and NQO1 (ab80588) were obtained from Abcam
(Cambridge, MA, USA). The β-actin antibody (sc-47778) and
peroxidase-conjugated goat anti-rabbit IgG or anti-mouse IgG were
obtained from ZSGB-BIO (Beijing, China). Immuno-complexes were
visualized on a luminescent imaging workstation (6600; Tanon, Shanghai,
China), and electrochemiluminescence was detected with BeyoECL Plus
(Beyotime Institute of Biotechnology, Beijing, China). Proteins were
quantified using scanning densitometry with ImageJ software (National
Institutes of Health, USA). Three independent experiments were
performed.